Antisense EGFRAS Guanidinium Peptide Nucleic Acid (GPNA) Oligonucleotides as Antitumor Agents

ABSTRACT

A class of antisense agents having a distributed guanidinium peptide nucleic acids (GPNA) backbone which has excellent uptake into mammalian cells, can bind to the target DNA or RNA in a highly sequence specific manner and can resist nucleases and proteases both outside and inside the cell(s) of interest. In one embodiment, either systemic or intratumoral administration of antisense Epidermal Growth Factor Receptor (“EGFR”) GPNA oligonucleotides is believed to downmodulate EGFR levels, thus in turn to reduce head and neck squamous cell carcinoma tumor growth, which has been confirmed to date both in vitro and in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/858,571 filed Nov. 13, 2007, which is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to GPNA oligonucleotides, and in particular toEGFRAS GPNA oligonucleotides, which has utility in treating malignanttumors.

2. Description of Related Art

Among other cancers, head and neck squamous cell carcinoma (HNSCC) is anaggressive malignancy that is difficult to treat with conventionaltherapies. Despite significant advances over the past 3 decades, currenttreatment modalities including surgery, radio- and chemotherapy have notimproved five-year survival rates in HNSCC patients. Molecularsignatures of HNSCC—indeed, all—malignancies is a topic of great currentinterest.

The Epidermal Growth Factor Receptor (EGFR) has emerged as a promisingmolecular target in the past decade or so, and in particular EGFR isupregulated in the majority of HNSCC tumors, so it is not surprisingthat preclinical model inhibition of EGFR has resulted in tumorinhibition. Notably, increased EGFR levels in HNSCC tumors have beenassociated with advanced stage, large tumor size, invasion, decreasedsurvival and poor prognosis. Notwithstanding this preclinical promise,clinical trials using agents that inhibit EGFR activation and signalinghave demonstrated limited antitumor efficacy. Another popular approachto target EGFR is to downmodulate its expression levels. Antisense DNAsequences that bind target DNA or mRNA inhibiting transcription ortranslation are highly effective in inhibiting HNSCC. However, DNA basedagents are prone to nuclease degradation and hence have short half-livesin plasma, and RNA- and/or DNA-based antisense agents are notcell-permeable, necessitating the finding of a carrier or effectivemodification before the agent can pass the cell membrane. Heretofore,then, the dual or triple goals of enzymatic stability, cell-permeabilityand antitumor effects have not been achieved by methods and agents knownprior to the present invention.

Therefore, a need remains for a way to administer antisense agents suchas EGFR Antisense (EGFRAS)—whether as complete genes or asoligonucleotides—that are resistant to nucleases and proteases and thuscan be given to patients systemically, i.e., parenterally or via otherappropriate systemic administration, to combat HNSCC and othermalignancies for which such treatment is indicated.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is a class ofantisense agents having a guanidinium peptide nucleic acid (GPNA)backbone which has excellent uptake into mammalian cells, can bind tothe target DNA or RNA in a highly sequence specific manner and canresist nucleases and proteases both outside and inside the cell(s) ofinterest. In particular, either systemic or intratumoral administrationof EGFRAS-GPNA oligonucleotides is believed to downmodulate EGFR levels,thus in turn to reduce HNSCC tumor growth, and this has been confirmedto date both in vitro and in vivo. Systemic administration of theparticular improved antisense oligonucleotides of the present inventionhas already exhibited antitumor activity.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 is a line graph contrasting the efficacy of intraperitoneal (IP)injection of EGFRAS GPNA with the intratumoral (IT) administration ofboth EGFRAS GPNA and EGFR Sense oligonucleotide control, in mice.

FIG. 2 is a line graph contrasting systemic delivery of EGFRAS GPNA andEGFR “scrambled;” in mice.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The present invention is a class of antisense agents having a peptidebackbone, called guanidinium peptide nucleic acids (GPNA), which canbind to the target DNA or RNA in a highly sequence specific manner whileevincing resistance to nucleases and proteases. In particular, eithersystemic or intratumoral administration of EGFRAS-GPNA oligonucleotidesis believed to downmodulate EGFR levels, thus in turn to reduce HNSCCtumor growth, and this has been confirmed to date both in vitro and invivo.

The present invention realizes the power of PNAs and PTDs, defined andexplained below, in a new and particular way. Peptide Nucleic Acids(PNAs) were known—prior to the present invention—for use as moieties inassociation with oligonucleotides in order to make the oligonucleotidesmore stable. Not only were PNAs already known to impart thisstabilization to oligonucleotides—PNAs were believed to have beeninvented in Denmark circa 1991—it was also believed prior to the presentinvention that particular PNAs could at least theoretically be used toimprove the uptake of PNAs into the cells (i.e., tissues such as tumortissues) of interest. For example, it was believed that by adding aProtein Transduction Domain (PTD) to a PNA, the oligonucleotide-PNA-PTDcombination would have increased uptake into cells. Previously knownPTDs included one or more arginine residues with a guanidinium portionthat rendered the resultant molecule amphipathic and concomitantly moreable to pass through cell membranes than non-amphipathic molecules.Unfortunately, these known PTDs were also unacceptably toxic to cells,as a general proposition. Therefore, as a part of the present inventionthe PTD of the PNA of the present oligonucleotides is positioned suchthat the PTD is protected by the PNA—by positioning the PTD within thePNA. This positioning of the PTD within the PNA increased the meltingpoint of the PNA/PTD/oligonucleotides to as high as 80° C. and increasedthe lipophilicity of the PNA/PTD/oligonucleotides such that not only didalmost 100% of the oligonucleotides enter the cells without the need foran additional carrier, such as a liposome, but the cell viabilityremained nearly 100% as well. The present invention, then, is generallyany oligonucleotide derivative having a PTD located within theassociated PNA (see the below descriptions of distributed and/oralternating guanidinium residues), and specifically the presentinvention embraces at least one particular EGFRAS GPNA oligonucleotidebelieved to cause new and unexpected therapeutic results whenadministered—particularly to patients having HNSCC tumors—eitherintratumorally or, almost unbelievably, systemically.

Those skilled in the art know, at this writing, that EGFR inhibition maybe achieved by either blocking EGFR autophosphorylation or bydownmodulating total EGFR levels within the cells of interest.Monoclonal antibodies and tyrosine kinase inhibitors that block EGFRhave demonstrated limited antitumor effects in clinical trials,unfortunately. This limited efficacy may be due to several reasonsincluding but not limited to rapid receptor turnover and inefficientblockade of the overexpressed receptor. Receptor downmodulation maycircumvent the problem of inefficient receptor inactivation. Previousreports demonstrate that EGFR antisense gene therapy can effectivelydownmodulate EGFR expression reducing HNSCC tumor cell viability.Preliminary results of a Phase I clinical trial in HNSCC patientsindicate a ˜430% clinical response rate with intratumoral delivery of anEGFR antisense gene. Although gene therapy cannot be deliveredsystemically, antisense oligonucleotides have proven feasible in cancerpatients when the oligonucleotides are administered intratumorally. Theadministration of antisense oligonucleotides has been limited, in theprior art, to intratumoral administration at least in part because priorart antisense oligonucleotides are rapidly degraded by serum nucleasesand because traditional PNAs having PTDs were too toxic to use.

The present GPNA oligonucleotides contain in part PNAs which, in detail,are DNA analogues in which the sugar-phosphate backbone is replaced by apseudopeptide chain of N-(2-aminoethyl) glycine monomers covalentlybonded to DNA bases. Nonionic PNAs form highly stable duplexes (throughWatson-Crick base-pairing to faun a hybrid) with complementary DNA andRNA strands and thus inhibit replication, transcription and translationof the template. The ability of PNAs to resist proteases and nucleases,and to hybridize stably, make PNAs attractive tools for biotechnologyapplications. Unfortunately, despite their neutral charge on thebackbone, PNAs are not readily taken up by mammalian cells and, asdescribed above, known PNAs with PTDs have been recognized as too toxicfor therapeutic use, so without improvements in PNAs their adoption hasbeen and would have continued to be extremely limited. In this context,then, the inclusion of the below-described internally linked D-arginineside chains is critical to the invention, and preferably the guanidineresidues are distributed (and more preferably alternatively distributedwith unmodified PNAs) throughout the oligonucleotide of interest.

The GPNAs of the present invention contain internally-linked D-arginineside chains (guanidinium residues) and bind to RNA with high affinityand sequence selectivity—and are readily taken up by mammalian cells.The present GPNAs, particularly the EGFRAS GPNAs, are believed to bindto the transcriptional start site of the EGFR gene to induce potent andsequence-specific antisense effects, in a manner less toxic than with amore traditional PNA-polyarginine conjugate.

In a manner typical of other syntheses according to the invention, GPNAmonomers have been synthesized according to an established solid-phaseBoc chemistry protocol. EGFRAS GPNA oligomers were synthesized in threesteps: 1) removal of the Boc-protecting group from the terminal amine;2) coupling of the next GPNA monomer onto the N-terminus of the growingchain and 3) capping of the unreacted amines with acetic anhydride.Following the complete coupling of the last monomer, GPNA were cleavedfrom the resins and the protecting groups were removed from thenucleobases and the guanidine group. The crude products wereprecipitated with ethyl ether and purified by reverse-phase HPLC. Theresulting EGFRAS GPNA were resuspended in water and characterized byMALDI-TOF mass spectrometry. In order to determine the efficiency ofuptake of the GPNA in HNSCC cells, fluorescently tagged EGFRAS GPNA wasalso synthesized, namely, a 16-base GPNA spanning nucleotides 764-779 onthe EGFR mRNA. On binding to its target, the C terminus of the GPNAA_(R)GC_(R)AG_(R)CT_(R)CC_(R)CA_(R)TT_(R)GG_(R)G (SEQ ID NO:1) binds tothe 5′ end of the mRNA or DNA targeted within the cell(s) to be treated.Alternative sequences could be, without limitation, those which targetthe transcriptional start site, such as,

T_(R)CG_(R)GG_(R)GA_(R)GC_(R)AG_(R)CG_(R)AT_(R)GC_(R)GA_(R)CC_(R)C (SEQID NO:2);or those which target the translational start site, such as

G_(R)GT_(R)CG_(R)CA_(R)TC_(R)GC_(R)TG_(R)CT_(R)C (SEQ ID NO:3),C_(R)GC_(R)AT_(R)CG_(R)CT_(R)GC_(R)TC (SEQ ID NO:4),G_(R)TC_(R)GC_(R)AT_(R)CG_(R)CT_(R)GC_(R)T (SEQ ID NO:5),G_(R)CA_(R)TC_(R)GC_(R)TG_(R)CT_(R)CC_(R)CC_(R)G (SEQ ID NO:6);

or additional alternative sequences including but not limited to

AG_(R)CA_(R)GC_(R)TC_(R)CC_(R)AT_(R)TG_(R)GG (SEQ ID NO:7),C_(R)CT_(R)CC_(R)GT_(R)GG_(R)TC_(R)AT_(R)GC_(R)TC_(R)C (SEQ ID NO:8),

C_(R)CC_(R)CA_(R)GC_(R)AG_(R)CT_(R)CC_(R)CA_(R)TT_(R)GG_(R)G (SEQ IDNO:9),

C_(R)GG_(R)AG_(R)GG_(R)TC_(R)GC_(R)AT_(R)CG_(R)CT_(R)G (SEQ ID NO:10).

The R groups indicate the guanidinium substituents, namely,N-(2-aminoethyl)D-arginine. In theory, although applicants do not intendto be bound by the theory, the distribution of the guanidinium residuesthroughout the oligonucleotide creates a conformational effect whichcontributes to lower toxicity of the oligonucleotide than whenguanidinium groups are provided to oligonucleotides in a less evenlydistributed fashion. It should be noted in the above GPNA sequences thateach nucleotide base without R residues are substituted with unmodifiedPNA, namely, N-(2-aminoethyl) glycine, such that the present GPNAs areindeed true PNAs even though every nucleotide base is notguanidinium-substituted. In the preferred embodiment of the presentinvention, the unmodified PNA and arginine-derived GPNA substitutionsalternate at every other nucleotide base position. However, alternateembodiments of the invention embrace both greater and lesserguanidinium-substitution, namely, at every nucleotide base position, atevery third, fourth or fifth nucleotide base substitution or any regularor irregular pattern of guanidinium substitution in the ratio of from1:1 nucleotide base: guanidinium group to 5:1 nucleotide base:guanidinium group, because as few as four guanidinium groups peroligomer can enhance uptake into the cells in contrast to oligomershaving no guanidinium substitution.

The invention is described with special particularity in the followingExamples.

Example 1

In vivo antitumor efficacy of EGFR antisense GPNA: As shown in FIG. 1,in order to examine the antitumor efficacy of EGFRAS GPNA(A_(R)GC_(R)AG_(R)CT_(R)CC_(R)CA_(R)TT_(R)GG_(R)G (SEQ ID NO: 1)) invivo a preliminary study was carried out in which athymic nude mice wereinoculated with head and neck squamous cell carcinoma (HNSCC) cell line1483 (10⁶ cells per site). Tumors were allowed to establish for 10 days.The tumor volumes were measured using a vernier caliper. Mice wererandomized into three groups such that the average of the tumor volumesin each group was the same. Two mice were treated with intraperitoneal(IP) injections of EGFRAS GPNA. Three mice were treated withintratumoral (IT) injections of EGFRAS GPNA and two mice were treatedwith EGFR sense oligonucleotide as a control. The mice were injectedonce per day for six days per week. Mice were administered 50 microgramsEGFRAS GPNA or EGFR sense oligonucleotide IT and 100 micrograms EGFRASGPNA IP. Treatment was carried out for 16 days. Tumors were measuredtwice a week in 2 dimensions using a vernier caliper. Tumor volumes wereestimated using the formula length×width (smaller dimension)/2. Theresults of the study were as follows. Mice treated with EGFRAS GPNA ITor IP had smaller tumors compared to the control EGFR senseoligonucleotides treated mice. There was no significant differencebetween tumor volumes of IT and IP treated EGFRAS GPNA groups,indicating that IP administration of GPNA is a feasible route ofdelivery. There was no apparently toxicity observed due to EGFRAS GPNAtreatment.

Example 2

Systemic delivery of EGFRAS GPNA has specific antitumor effects in HNSCCxenografts: Referring now to FIG. 2, athymic nude mice were inoculatedwith HNSCC cell line 1483 subcutaneously with one million cells perflank. Animals were randomized into 3 groups based up on the tumorvolumes. There were 11 mice in the EGFRAS GPNA group and 10 mice each inthe saline and the EGFR scrambled groups. Saline or GPNA (5 mg/kg bodyweight) were administered via IP injections 5 days a week. Treatment wascarried out for 10 days. Tumors were measured twice a week in 2dimensions using a vernier caliper. Tumor volumes were estimated usingthe formula length×width (smaller dimension)/2. There was a significantdifference in the tumor volumes of mice treated with EGFR antisense GPNAcompared to the scrambled GPNA (P<0.01). There was no significantdifference between the tumor volumes of mice treated with the scrambledGPNA and saline.

1. A method for the treatment of malignancy comprising administering systemically or intratumorally an oligonucleotide comprising at least one peptide nucleic acid “PNA”, the peptide nucleic acid comprising a guanidinium peptide (GPNA), to a patient in need of such treatment, further wherein said oligonucleotide consists of a 13-22 base GPNA spanning all or a portion of nucleotides 758-785 on an EGFR mRNA wherein said 13-22 base GPNA has both unmodified PNA and four or more arginine-derived GPNA substitutions.
 2. The method according to claim 1 wherein said oligonucleotide has a guanidinium residue substitution of 4-22.
 3. The method according to claim 1 wherein said 13-22 base GPNA has unmodified PNA and arginine-derived GPNA substitutions alternating at every other nucleotide base position and numbering between 6 and
 11. 4. A peptide nucleic acid “PNA” comprising a guanidinium peptide (GPNA) comprising an oligonucleotide consisting of a 13-22 base GPNA spanning all or a portion of nucleotides 758-785 on an EGFR mRNA wherein said 13-22 base GPNA has both unmodified PNA and four or more arginine-derived GPNA substitutions.
 5. The peptide nucleic acid according to claim 4 wherein said oligonucleotide has a guanidinium residue substitution of 4-22.
 6. The peptide nucleic acid according to claim 4 wherein said 13-22 base GPNA has unmodified PNA and arginine-derived GPNA substitutions alternating at every other nucleotide base position and numbering between 6 and
 11. 